Yeast Rnt1p is required for cleavage of the pre-ribosomal RNA in the 3 ' ETS but not the 5 ' ETS

We have reexamined the role of yeast RNase III(Rnt1p) in ribosome synthesis . Analysis of pre-rRNA processing in a strain carrying a complete deletion of the RNT1 gene demonstrated that the absence of Rnt1p does not block clea vage at site A(0) in the 5' external transcribed spacers (ETS), although th e early pre-rRNA cleavages at sites A(0), A(1), and A(2) are kinetically de layed. In contrast, cleavage in the 3' ETS is completely inhibited in the a bsence of Rnt1p, leading to the synthesis of a reduced level of a 3' extend ed form of the 25S rRNA. The 3' extended forms of the pre-rRNAs are consist ent with the major termination at site T2 (+210). We conclude that Rnt1p is required for cleavage in the 3' ETS but not for cleavage at site Ao. The s ites of in vivo cleavage in the 3' ETS were mapped by primer extension. Two sites of Rnt1p-dependent cleavage were identified that lie on opposite sid es of a predicted stem loop structure, at +14 and +49. These are in good ag reement with the consensus Rnt1p cleavage site. Processing of the 3' end of the mature 25S rRNA sequence in wild-type cells was found to occur concomi tantly with processing of the 5' end of the 5.8S rRNA, supporting previous proposals that processing in ITS1 and the 3' ETS is coupled.